J. Dairy Sci.
104
https://doi.org/10.3168/jds.2021-20486
© 2021 American Dairy Science Association®. Published by Elsevier Inc. and Fass Inc. All rights reserved.
Prepartum level of dietary cation-anion difference fed to nulliparous
cows: Acid-base balance, mineral metabolism, and health responses
R. Zimpel,1,2 M. Nehme Marinho,1 K. V. Almeida,1 A. Revilla Ruiz,1 M. C. Perdomo,1 M. B. Poindexter,1
A. Vieira-Neto,1,2 U. Arshad,1,2 A. Husnain,1,2 C. D. Nelson,1,2 and J. E. P. Santos1,2*
1
Department of Animal Sciences, University of Florida, Gainesville 32611
2
DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville 32611
ABSTRACT
Objectives were to determine the effects of 3 different
levels of dietary cation-anion difference (DCAD) fed
during the last 22 d of gestation to pregnant nullipa-
rous cows on pre- and postpartum acid-base balance,
mineral metabolism, and health responses. In all, 132
pregnant nulliparous Holstein cows were enrolled at 250
(248–253) d of gestation, blocked by genomic merit of
energy-corrected milk yield, and assigned randomly to
diets varying in DCAD: +200 (P200, n = 43), −50
(N50, n = 45), or −150 (N150, n = 44) mEq/kg of dry
matter. Dietary treatments were fed until calving, after
which cows received the same lactation diet for the
first 100 d postpartum. Urine and blood were sampled
throughout the prepartum period and in the first weeks
postpartum, and urine was assessed for pH, whereas
blood was analyzed for gases, measures of acid-base
balance, minerals, and metabolites. Calcium (Ca) and
magnesium (Mg) retention and phosphorus (P) digest-
ibility were evaluated in the last week of gestation and
first week of lactation. Incidence of diseases was evalu-
ated for the first 100 d postpartum. Data are presented
in sequence as P200, N50, N150 (LSM ± SEM). Reduc-
ing the DCAD reduced urine (8.17 vs. 6.50 vs. 5.51 ±
0.11) and blood pH (7.442 vs. 7.431 vs. 7.410 ± 0.004)
and induced a state of compensated metabolic acidosis
with a reduction in blood HCO3- (28.4 vs. 26.7 vs. 24.9
± 0.3 mM) and partial pressure of CO2 (41.8 vs. 40.1
vs. 39.1 ± 0.4 mmHg) prepartum. Reducing the DCAD
linearly increased blood ionized Ca (iCa; 1.224 vs. 1.243
vs. 1.259 ± 0.008 mM) and serum total Ca (tCa; 2.50
vs. 2.53 vs. 2.56 ± 0.02 mM) prepartum, blood iCa
on the day of calving, and serum Mg in the first days
Received March 18, 2021.
Accepted July 18, 2021.
*Corresponding author: jepsantos@​ufl​.edu
postpartum. Reducing the DCAD linearly increased
the apparent absorption of Ca (12.9 vs. 19.0 vs. 20.9 ±
1.4 g/d) and Mg (7.0 vs. 9.9 vs. 10.4 ± 1.4 g/d) prepar-
tum, but apparent retention of both Ca (13.9 g/d) and
Mg (3.4 g/d) did not differ with treatment. Treatment
did not affect digestibility of P pre- or postpartum
or retention of Ca or Mg postpartum. Treatment did
not affect the incidence or prevalence of subclinical
hypocalcemia, hepatic composition, or the prevalence
of fatty liver. Reducing the DCAD had a quadratic
effect on incidence of fever (46.5 vs. 17.6 vs. 33.9 ±
7.0%), uterine diseases (36.3 vs. 25.6 vs. 46.0 ± 7.3%),
and morbidity (41.4 vs. 28.1 vs. 55.6 ± 7.3%). Feed-
ing a diet with −50 mEq/kg of dry matter promoted
moderate changes in acid-base balance, altered mineral
metabolism, and benefited health of nulliparous cows;
however, further reducing the DCAD to −150 mEq/kg
negated the benefits to health.
Key words: dietary cation-anion difference, health,
mineral retention, nulliparous cow
INTRODUCTION
Hypocalcemia is often studied in dairy cows start-
ing their second or greater lactation, and evidence ex-
ists for changes in mineral metabolism as animals age
(Horst et al., 1990). Concentrations of total Ca (tCa)
and ionized Ca (iCa) are greater in calves than adult
cows, likely because the set point for controlling iCa
concentration in blood changes as the animal ages. In
rats, Ca-sensing receptor mRNA and protein expres-
sion in numerous tissues change with age (Autry et
al., 1997). Young animals are continuously accreting
bone as they grow, and bone density increases with
age (Jones et al., 1978). Evidence exists for greater
bone remodeling in primiparous than multiparous cows
(Rodney et al., 2018). Furthermore, primiparous cows
experience a smaller Ca loss as colostrum than mul-
tiparous cows because of the reduced colostrum yield
(Rodney et al., 2018). Thus, it is not surprising that
 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS
milk fever is seldom diagnosed in primiparous cows, as
they maintain greater concentrations of iCa and tCa at
the onset of lactation compared with multiparous cows
(Rodney et al., 2018). Because of that, the incidence or
prevalence of subclinical hypocalcemia is less than half
of that observed in multiparous cows (Reinhardt et al.,
2011; Neves et al., 2018).
Acidogenic diets are widely fed prepartum to prevent
milk fever in dairy cows. Mineral metabolism, in par-
ticular Ca and, to a lesser extent, P and Mg, is altered
with the onset of lactation because of extensive irrevers-
ible loss to synthesize colostrum and milk (Rodney et
al., 2018). At the same time that mineral losses increase
with synthesis of milk, the onset of lactation results
in homeorhetic adaptations resulting in an increase in
gastrointestinal Ca absorption and a decrease in Ca
flux to bones and urinary and fecal losses (Ramberg et
al., 1970). Although nulliparous cows represent a large
proportion of the transition cows on a farm, limited
research exists with manipulating the DCAD in diets
fed to prepartum nulliparous cows. Two recent meta-
analyses detected an interaction between prepartum
DCAD and parity group for yield of FCM because the
benefits of reducing the DCAD to productive perfor-
mance were only observed in parous cows (Lean et al.,
2019; Santos et al., 2019). Nevertheless, the authors
did not observe the same interaction between DCAD
and parity group for risk of diseases, implying that the
reduced risk of retained placenta and metritis observed
in the meta-analysis applied to both nulliparous and
parous cows (Lean et al., 2019; Santos et al., 2019).
Nulliparous cows might benefit from manipulation of
the DCAD prepartum, but perhaps diets should be less
acidogenic than those typically fed to parous cows to
prevent milk fever.
We hypothesized that feeding a diet with intermedi-
ate negative DCAD prepartum would improve mineral
metabolism in nulliparous cows during the pre- and
postpartum periods, which would benefit health in
early lactation. Thus, objectives of the experiment were
to determine the effects of 3 different levels of DCAD
in prepartum diets fed to nulliparous cows on pre- and
postpartum mineral metabolism and health responses
in early lactation.
MATERIALS AND METHODS
The University of Florida Institutional Animal Care
and Use Committee approved all procedures involv-
ing cows in the experiment under protocol number
201810505. The experiment was conducted at Univer-
sity of Florida Dairy Unit, Gainesville, between Novem-
ber 2018 and November 2019.
Journal of Dairy Science Vol. 104 No. 12, 2021
Sample Size Calculation, Experimental Design,
and Dietary Treatments
A sample size was calculated using the POWER pro-
cedure of SAS (SAS/STAT version 9.4; SAS Institute
Inc.) based on productive responses detailed in Zimpel
et al. (2021). The a priori sample size was calculated
based on yield of ECM, although the experiment evalu-
ated numerous other responses of interest.
The experiment followed a randomized complete
block design. Cohorts of 3 to 13 nulliparous cows were
ranked by genomic merit for yield of ECM, and each
3 cows were assigned a block. Within each block, cows
were assigned randomly to 1 of 3 dietary treatments
that were fed as TMR that varied in DCAD. Treat-
ments were +200 (P200; n = 43), −50 (N50, n = 45),
or −150 (N150, n = 44) mEq/kg of DM. Cows enrolled
in the experiment were lactation 0 prepartum and lac-
tation 1 postpartum. For consistency, throughout the
manuscript cows will be designated as nulliparous.
Dietary treatments were applied only during the
prepartum period and changes in DCAD were achieved
by manipulating ingredients in the diet to increase Cl
and S contents and adjust other minerals to equiva-
lent concentrations in all 3 treatments. The DCAD
was calculated using the following formula: DCAD
= [(mEq of K + mEq Na) − (mEq Cl + mEq S)].
The DCAD was manipulated by incorporating an ac-
idogenic product (Bio-Chlor; Arm & Hammer Animal
and Food Production) containing dried condensed
extracted glutamic acid fermentation product, dried
condensed corn fermentation solubles, processed grain
by-products, and magnesium chloride. The acidogenic
product was incorporated into the diet at increasing
proportions to replace citrus pulp and expeller soybean
meal (Supplemental Table S1, https:​/​/​figshare​.com/​s/​
23982480396e0155e6bd). Treatment diets were fed as
TMR starting at 250 d of gestation until calving, and
the mean and median days in the treatment diets were,
respectively, 21.6 and 21 d for P200, 22.9 and 22 d for
N50, and 22.1 and 22 for N150. Upon calving, cows
were fed the same lactation diet as a TMR for the
first 100 d postpartum. The nutrient composition of
dietary treatments is presented in Supplemental Table
S2 (https:​/​/​figshare​.com/​s/​23982480396e0155e6bd).
Details of sampling and chemical analyses of ingredient
and diets are presented in Zimpel et al. (2021).
Prepartum cows were fed for ad libitum intake once
daily, at 0800 h. Postpartum cows were fed for ad libi-
tum intake twice daily, at 0630 and 1130 h. Individual
feed intake was measured daily during the pre- and
postpartum periods. The amounts of feed offered to
individual cows were adjusted daily to result in at least
 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS
8% refusals, which were weighed once daily, before the
morning feeding. Investigators were not blind to treat-
ments.
Urine Collection and Analyses of pH
Urine was sampled 3 times weekly starting on d 251
of gestation until calving, by manually stimulating
the perineal area until obtaining a clean and copious
stream. Approximately 50 mL of urine was sampled
and had pH measured within 30 min of collection (Ac-
cumet AR15 pH meter, Fisher Scientific International
Inc.). Urine was also sampled, and pH measured on d
270 and 271 of gestation from a subset randomly se-
lected blocks that had 23, 26, and 26 cows in the P200,
N50, and N150 treatment, respectively, to characterize
the pattern of urine pH throughout the day. Each day,
samples were collected at intervals of 6 h, but advanc-
ing 3 h the sampling time on the second day to result
in sampling times with 3 h intervals relative to feeding
time, thus representing 0, 3, 6, 9, 12, 15, 18, and 21 h
relative to feeding time.
Sampling Whole Blood and Measurements
of Acid-Base Status
Whole blood was sampled by puncture of the jugular
vein into 10 mL lithium-heparinized tubes (Vacutainer
system, Becton, Dickinson Co.) on d 268 and 271 of
gestation from a subset randomly selected blocks that
had 28, 32, and 31 cows in the P200, N50, and N150
treatment, respectively, which corresponded to days −6
and −3 relative to calving in the sampled cows. Blood
also was sampled from a subset of randomly selected
blocks that had 34, 35, and 36 cows in the P200, N50,
and N150 treatment, respectively, on d 0, immediately
after calving, and at 1, 2, and 4 d postpartum. Whole
blood was analyzed within 1 min of sampling using
a hand-held biochemical analyzer (VetScan i-STAT,
Abaxis). Analytes quantified included pH; blood gases,
including partial pressure CO2 (pCO2), partial pres-
sure of O2 (pO2), total dissolved CO2 (TCO2), and
saturation of O2 (sO2); minerals, including iCa, ionized
Na (iNa), and ionized K (iK); and concentrations of
HCO3-, base excess, hematocrit, and hemoglobin.
Blood Sampling and Processing
All blood samplings for serum separation were per-
formed early in the morning before feeding, except for
the day of calving, in which blood was collected in the
first 41 ± 47 min (mean ± SD) after parturition re-
gardless of the time of the day. Starting on d 264 of
Journal of Dairy Science Vol. 104 No. 12, 2021
gestation, blood was sampled from all cows 4 times/
wk on Monday, Wednesday, Friday, and Saturday, until
calving, and then on the day of calving and on d 1, 2,
3, 4, 5, 7, 14, and 21 postpartum by puncture of the
coccygeal vessels into 10-mL serum separator evacu-
ated tubes (Vacutainer system, Becton Dickinson Co.).
Blood was allowed to clot for 30 min, then tubes were
placed in ice and transported to the laboratory within
1 h of collection. Tubes were centrifuged for 20 min at
2,000 × g at room temperature for serum separation.
Serum samples were transferred into multiple aliquots
of 1.5 mL and stored frozen at −20°C until analyses.
Blood Measurements
Serum samples collected on d −9, −7, −5, −3, −1, 0,
1, 2, 3, 4, 5, and 7 relative to calving were analyzed for
concentrations of tCa, tP, tMg. Samples collected on
d −7, 0, 1, 2, 3, 4, 5, 7, 14, and 21 relative to calving
were analyzed for concentrations of glucose. Samples
collected on d −7, 0, 7, 14, and 21 relative to calving
were analyzed for concentrations of fatty acids, and
those collected on d 7, 14, and 21 were analyzed for
concentrations of BHB. All assays followed the initial
randomization with blocks, such that samples from a
given block were analyzed in the same assay.
Concentrations of tCa and tMg were analyzed by
atomic absorption using a spectrophotometer equipped
with Ca- and Mg-specific hollow cathode lamps (AAna-
lyst 200, Perkin-Elmer Inc.) as described by Martinez
et al. (2012). Intra- and interassay coefficients of varia-
tion (CV) were 3.7 and 3.7%, respectively, for tCa and
2.8 and 5.4%, respectively, for tMg. Concentrations of
tP were analyzed using the molybdenum blue method
(Quinlan and DeSesa, 1955). The intra- and interassay
CV were 3.6 and 7.2%, respectively. Concentrations of
fatty acids (NEFA-C kit; Wako Diagnostics Inc.) ac-
cording to Johnson and Peters (1993) and BHB (Wako
Autokit 3-HB; Wako Diagnostics Inc.) were analyzed
using colorimetric enzymatic assays. Intra- and interas-
say CV were 2.8 and 8.1%, respectively, for fatty acids,
and 2.7 and 5.0%, respectively, for BHB. Concentra-
tions of glucose were analyzed using a colorimetric as-
say (TECO Diagnostic). Intra- and interassay CV were
5.1 and 8.2%, respectively.
Sampling for Mineral Retention
Prepartum mineral retention was determined from
a subset of 26, 30, and 29 cows in the P200, N50, and
N150 treatment, respectively. The subset represented
cows that calved after 271 d of gestation such that
samples from both days could be used for laboratory
 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS
assays. Fecal grab samples were collected through tran-
srectal palpation, approximately 200 g each, at 6 h in-
tervals on d 270 and 271, advancing 3 h on d 271 such
that samples collected would represent 0, 3, 6, 9, 12, 15,
18, and 21 h relative to feeding. Total mixed diets and
orts were collected from each cow immediately before
feeding on d 268, 269, 270, and 271 of gestation. Orts,
TMR, and fecal samples were dried in an air-forced
oven at 60°C for 48 h and stored until analyses. Urine
samples were collected concurrent with samples as pre-
viously described. Samples were frozen at −20°C until
analyses.
Postpartum mineral retention was determined in a
subset of 35, 39, and 34 cows in the P200, N50, and
N150 treatment, respectively. Fecal and urine samples
were collected on d 5 and 6 postpartum exactly as de-
scribed for the prepartum period to represent a 24-h
feeding cycle. Diets and orts were sampled from in-
dividual cows on d 3, 4, 5, and 6 postpartum. Milk
samples were collected from 0630 and 1830h milkings
on d 1, 2, 3, 4, and 5 d postpartum and frozen at −20°C
until analyses. The concentrations of minerals and the
yield of milk on d 3 to 5 were used for calculations of
mineral retention postpartum.
For the pre- and postpartum periods, TMR, orts, and
fecal samples were ground to pass a 1-mm screen of a
Wiley mill (Thomas Scientific). Dried TMR, orts, and
fecal samples from individual cows were composited for
the prepartum and postpartum periods before chemical
analyses. Urine samples referring to the pre- and post-
partum periods for each cow were composited in one
sample prepartum and one sample postpartum before
analyses. Milk samples were analyzed individually and
those from d 3, 4, and 5 were used for mineral retention
postpartum.
Analyses of Samples and Calculations
of Mineral Retention
Dried ground TMR, orts, and fecal samples were
analyzed for DM at 105°C for 24 h, then 2.00 ± 0.05
g of samples were placed in previously dried crucibles
and heated at 600°C for 8 h, and ash content was deter-
mined. Ten milliliters of a 3N solution of HCl was added
to each crucible, covered, and boiled on a hot plate for
10 min for acid digestion. Crucibles were rinsed with
deionized water and then samples were filtered into a
100-mL volumetric flasks that were filled with deion-
ized water to reach 100 mL. Samples were then vigor-
ously mixed and further diluted 1:20 for Ca and 1:400
for Mg analysis with 1% lanthanum solution (117.3 g of
lanthanum oxide diluted into 500 mL of 3N HCl, and
further diluted to reach 10 L using deionized water)
Journal of Dairy Science Vol. 104 No. 12, 2021
or diluted to 1:2 with deionized water for P analyses.
Concentrations of Ca and Mg were analyzed by atomic
absorption using a spectrophotometer (AAnalyst 200;
Perkin-Elmer Inc.) equipped with a hollow cathode
lamp to detect Ca and Mg. Intra- and interassay CV
were, respectively, 3.5 and 4.4% for Ca, and 5.9 and
3.1% for Mg. Concentrations of P were analyzed using
the molybdenum blue method (Quinlan and DeSesa,
1955). Intra- and interassay CV were, respectively, 2.3
and 3.9%. All procedures were performed in duplicates.
Urine samples were analyzed for Ca, Mg, and creati-
nine. Samples were diluted 1:400 with 0.5% lanthanum
chloride and concentrations of Ca and Mg were ana-
lyzed by atomic absorption. Samples were analyzed in
duplicate. Intra- and interassay CV were, respectively,
5.4 and 4.8% for Ca, and 2.7 and 2.3% for Mg. Urine
samples were analyzed for creatinine using a commer-
cial colorimetric method (Creatinine Urinary Detection
Kit; Arbor Assays). Samples were analyzed in dupli-
cate. Intra- and interassay CV were, respectively, 1.0
and 2.8%. Creatinine was used as a marker to estimate
daily urinary volume based on the constant excretion of
29 mg of creatinine/kg of BW per day (Valadares et al.,
1999). The estimate of daily urinary volume was calcu-
lated using the mean BW of each cow in the week the
urine sample was taken. Calculation of urinary volume
was performed as follows: Urine (L/d) = (BW × 29)/
creatinine concentration in urine (mg/L). Daily urinary
excretions of Ca and Mg were calculated as the product
of urinary volume and the respective concentrations of
those minerals in the urine samples.
Concentrations of Ca and Mg in milk were analyzed
in individual milk samples by atomic absorption as
described by Vieira-Neto et al. (2017). Samples were
analyzed in duplicate. Intra- and interassay CV were,
respectively, 1.5 and 3.2% for Ca and 1.8 and 3.3% for
Mg. Milk yield from each shift of sampling (i.e., 0030
and 0630 h representing the morning shift, and 1230
and 1830h representing the afternoon shift) was used to
calculate the final concentrations of Ca and Mg in milk.
Daily milk secretion of Ca and Mg were calculated as
the product of milk yield and the respective concentra-
tions of those minerals in the milk samples.
Indigestible NDF (iNDF) was used to estimate total
fecal output and samples of TMR, feces, and orts sam-
ples were analyzed for iNDF. Samples were prepared
in quadruplicates in nylon bags with a pore size of 25
μm (Ankom Technology), with 2 samples from each
specimen incubated in the rumen of 2 cannulated cows
for 288 h for in situ digestion as previously described
(Huhtanen et al., 1994). Following in situ incubation,
bags were retrieved, and the residue analyzed for NDF
(Van Soest et al., 1991). The means for intraassay with-
 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS
in and between cows were 4.2 and 6.1%, respectively.
The iNDF intake was calculated as: iNDF intake, kg/d
= (DM offered × concentrations of iNDF in the diet
offered) − (DM refused × iNDF in the refusal). Fecal
output was calculated as Feces, kg/d = iNDF intake/
concentration of iNDF in feces. Apparent digestibility
of Ca, P, and Mg were calculated based on intake of the
respective minerals and their fecal excretions. Reten-
tions of Ca and Mg prepartum were calculated based on
intake minus fecal and urinary excretions. Retention of
Ca and Mg postpartum were calculated based on intake
minus fecal and urinary excretion and milk secretion.
Liver Tissue Collection
Liver tissue was collected once for biopsy on d 10
(9–11) postpartum. Briefly, the cow was restrained, and
areas between the 11th and 13th right intercostal spac-
es were scanned by ultrasonography (Aloka SSD-500V
equipped with a 3.5-MHz convex transducer, Aloka Co.
Ltd.) to determine the location for sampling hepatic
tissue. The area was shaved and thoroughly cleansed
with 3 alternating rounds povidone-iodine scrubbing
followed by a 70% isopropyl alcohol rinse. Upon disin-
fecting the surgical site, 15 mL of a solution containing
2% lidocaine hydrochloride was administered in the
subcutaneous and intramuscular spaces. The surgical
site was disinfected again as described previously. After
a 2-cm incision of the skin, a stainless-steel percutane-
ous hepatic biopsy tool (Aries Surgical) was introduced
aiming the hepatic tissue and approximately 1 g of tis-
sue was collected. The tissue was placed in filter paper,
rinsed with sterile saline, sliced into 3 sections, and
transferred into separate cryovials, snap-frozen in liq-
uid N, and stored at −80°C until analysis.
Analyses of Liver Tissue
Hepatic triacylglycerol was analyzed on liver sampled
from a subset of 33 blocks of cows containing 33, 33,
and 32 cows in P200, N50, and N150, respectively. One
cryovial containing at least 300 mg of hepatic tissue
was thawed on ice, and the sample transferred to fil-
ter paper to dry. A 300 mg sample was homogenized
in 3 mL of saline solution using an Ultra-Turrax T25
homogenizer (Rose Scientific Ltd.). A 6-mL solution of
1:1 saline and methanol was added to each sample and
then split into 2 equal aliquots. One aliquot was used
to analyze tissue DM and second aliquot was used to
analyze triacylglycerol. Dry matter was determined in
triplicate, each sample containing 0.5 mL dried at 55°C
for 48 h in a forced-air oven. A second aliquot was ana-
lyzed for concentration of triacylglycerol in triplicate.
Journal of Dairy Science Vol. 104 No. 12, 2021
Lipids were extracted in triplicate using a 2:1 solution
of chloroform and methanol (Folch et al., 1957). Each
of the 3 extracted samples were assayed for triacylg-
lycerol using a colorimetric method (Foster and Dunn,
1973). Intra- and interassay CV were, respectively, 6.3
and 11.7%. Cows were considered to have fatty liver
if hepatic triacylglycerol was greater than 5.0% of wet
tissue (Gaal et al., 1983).
Characterization and Diagnosis of Clinical
and Subclinical Diseases and Survival
Incidence of clinical diseases was evaluated from
calving to 100 d postpartum. No diagnosis of milk fever
occurred during the experiment, and no cow received
any oral, intravenous, or subcutaneous Ca therapy dur-
ing the experiment. The lack of diagnosis of milk fever
was later confirmed as the smallest tCa concentration
in serum was 1.47 mM. All cows underwent a complete
physical examination on d 4, 7, 10, and 12 postpartum.
In addition, any cow with altered behavior that might
indicate disease such as inappetence or a decline in
milk yield were also examined. Cows that had not shed
the placenta by 24 h after calving were considered to
have retained fetal membranes. Rectal temperature was
measured, between 0800 and 1000 h, on d 1, 2, 3, 4, 5, 7,
9, 11, 13, and 15 postpartum, and fever was considered
when temperature was greater than 39.5°C. Cows were
transrectally palpated and presence of an enlarged flac-
cid uterus with fetid watery reddish-brownish discharge
was defined as metritis (Sheldon et al., 2009). Cows
that had metritis concurrent with fever were considered
to have puerperal metritis. Diagnosis of displaced ab-
omasum was performed by percussion and auscultation
of the left and right cranial areas adjacent to the para-
lumbar fossa followed by confirmation during corrective
surgery. Pneumonia was diagnosed based on increased
respiratory frequency and presence of abnormal lung
sounds at auscultation concurrent with fever. Lameness
was diagnosed based on visual lameness score during
standing and walking followed by routine therapeutic
hoof trimming. Immediately before each milking, all
cows were examined for signs of clinical mastitis by
the herd personnel based on the presence of abnormal
milk in one or more quarters. Morbidity included cows
that had at least one of the following clinical diseases:
retained placenta, metritis, displaced abomasum, mas-
titis, lameness, or respiratory disease. Cows with more
than one clinical disease were classified as having mul-
tiple diseases. Survival of cows was evaluated until 305
d postpartum.
Incidence of subclinical hypocalcemia was based
on at least 1 sample with whole blood iCa ≤1.0 mM
 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS
(Oetzel et al.,1988) on d 0, 1, 2, and 4 postpartum, or
serum tCa ≤2.0 mM (Reinhardt et al., 2011) on d 0, 1,
2, 3, 4, 5, and 7 postpartum. Daily prevalence of sub-
clinical hypocalcemia, based on serum tCa ≤2.0 mM,
was evaluated on d 0, 1, 2, 3, 4, 5, and 7 postpartum.
Hyperketonemia was defined as serum BHB concentra-
tions >1.20 mM on at least one sample collected on d
7, 14, and 21 postpartum (McArt et al., 2011).
Statistical Analyses
Normality of residuals and homogeneity of variance
were examined for each continuous dependent variable
analyzed after fitting the statistical models. Responses
that violated the assumptions of normality were sub-
jected to power transformation according to the Box-
Cox procedure (Box and Cox, 1964) using a macro for
mixed models in SAS (Piepho, 2009). The LSM and
SEM were back transformed for presentation of results
according to Jørgensen and Pedersen (1998). Pre- and
postpartum data were analyzed separately.
Continuous data were analyzed by ANOVA with lin-
ear mixed-effects models using the MIXED procedure
of SAS. The statistical models included the fixed effects
of treatment (P200 vs. N50 vs. N150), time of measure-
ment, the interaction between treatment and time, and
the random effects of block and cow nested within treat-
ment. For prepartum data, the exact day relative to
calving was used as covariate in the statistical analyses.
Time was the term in the REPEATED statement and
the covariance structure was selected based on spac-
ing of measurements and model fit assessed based on
the smallest Akaike’s information criterion. For analy-
ses with unequal spacing, the spatial power was the
selected covariance structure. For responses analyzed
with equal spacing between measurements, first-order
autoregressive was the most commonly selected covari-
ance structure. Responses with a single measurement
per cow were analyzed with models that included the
fixed effect of treatment and the random effect of block.
When an interaction between treatment and time re-
sulted in P < 0.10, then means at different time points
were partitioned using the SLICE command of SAS.
Binary data were analyzed by generalized linear
mixed-effects models using logistic regression with the
GLIMMIX procedure of SAS fitting a binary distribu-
tion. The statistical models included the fixed effects of
treatment, calf sex (male vs. female), and the genomic
PTA value for the respective disease, and the random
effect of block. For incidence or prevalence of diseases,
the genomic predicted transmitting ability value for
the respective disease was used as covariate in the
statistical models. For binary data with repeated mea-
Journal of Dairy Science Vol. 104 No. 12, 2021
sures within cow such as the mean daily prevalence of
subclinical hypocalcemia, hyperketonemia, and fever,
the random effect of cow nested within treatment was
included in the mixed models. For analyses of incidence
of displaced abomasum and mastitis, Fisher’s exact
test was used because of the low frequency of events.
In all mixed-effects models, the Kenward-Roger
method was used to approximate the denominator de-
grees of freedom for the F tests. Orthogonal polynomial
contrasts were used to determine linear and quadratic
effects of treatments on the response analyzed. The
interactive matrix language procedure of SAS was used
to generate the orthogonal coefficients to adjust for the
unequal spacing of DCAD in treatments. The resulting
coefficients for P200, N50, and N150 were, respectively,
0.787, −0.206, and −0.581 for the linear contrast, and
0.217, −0.790, and 0.573 for the quadratic contrast.
Days to leave the herd was analyzed with the Cox’s
proportional hazard regression using the PHREG pro-
cedure of SAS. The model included the fixed effects
of treatment, calf sex, and the genomic PTA value for
productive life. The adjusted hazard ratios and respec-
tive 95% confidence interval were calculated. Survival
curves were generated adjusted for covariates in the
statistical models using the BASELINE statement in
PHREG of SAS. Model fit was assessed by plotting the
martingale and deviance residuals against the estimates
of the linear predictor. Proportionality of hazards among
treatments was evaluated using the ASSESS statement
for proportional hazard in PHREG. The median days
to leaving the herd were not computed because only a
small fraction of cows left the herd by 305 d postpar-
tum; thus, only the LSM ± SEM for days in the herd
were calculated with the LIFETEST procedure of SAS.
Treatment differences with P ≤ 0.05 were considered
significant, whereas tendencies for differences were re-
ported if 0.05 < P ≤ 0.10.
RESULTS
All 132 nulliparous cows enrolled in the experiment
contributed data for statistical analyses. Two cows
from treatment P200 died: one developed peritonitis
after calving and died 5 d postpartum, and one cow was
euthanized at 70 d postpartum because of a hip lesion.
Both cows contributed data until the day they left the
experiment, at 5 and 70 d postpartum. Supplemental Ta-
ble S3 (https:​/​/​figshare​.com/​s/​23982480396e0155e6bd)
depicts the characteristics of nulliparous cows enrolled
in the experiment according to treatment. There was
no evidence of difference among treatments for almost
all responses analyzed confirming proper balance of
cows among treatments.
 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS

Table 1. Effect of DCAD fed prepartum to nulliparous Holstein cows on prepartum measures of blood chemistry, acid-base balance, and urine
pH

Treatment2 P-value3

Item1 P200 N50 N150 SEM TRT × time Linear Quadratic

4
Venous blood gas              
 pCO2, mm Hg 41.8 40.1 39.1 0.5 0.12 <0.001 0.65
 pO2, mm Hg 33.1 33.4 33.1 0.6 0.19 0.92 0.65
 TCO2, mM 29.8 27.9 25.9 0.4 0.14 <0.001 0.06
 sO2, % 65.6 65.4 63.3 1.1 0.23 0.21 0.27
Venous blood chemistry              
 pH 7.442 7.431 7.410 0.004 0.42 <0.001 0.01
 HCO3−, mM 28.4 26.7 24.9 0.4 0.43 <0.001 0.05
  Base excess, mM 4.43 2.49 0.06 0.44 0.35 <0.001 0.01
  Hematocrit, % 28.5 27.7 28.2 0.4 0.94 0.39 0.23
  Hemoglobin, g/dL 9.71 9.41 9.59 0.15 0.90 0.36 0.23
Urine pH              
 Daily5 8.22 6.67 5.41 0.05 0.004 <0.001 <0.001
  Every 3 h6 8.17 6.50 5.51 0.11 0.71 <0.001 0.07
1
Blood sampled by puncture of the jugular vein on days −6 and −3 relative to calving from a subset of randomly selected blocks that had 28,
32, and 31 cows in the P200, N50, and N150 treatment, respectively.
2
Nulliparous cows were fed prepartum diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation
to calving; after calving, cows were fed the same lactation diet for the first 100 d postpartum.
3
TRT × time = interaction between treatment (P200 vs. N50 vs. N150) and time (day or hour); Linear = linear effect of DCAD; Quadratic =
quadratic effect of DCAD.
4
pCO2 = partial pressure of CO2; pO2 = partial pressure of O2; TCO2 = total dissolved CO2; sO2 = saturation of O2.
5
Urine sampled 3 times weekly from 251 d of gestation until calving.
6
Urine sampled every 3 h relative to the time of feeding on gestation d 270 and 271.

Prepartum Acid-Base Balance and Urinary pH
Reducing the DCAD induced a compensated meta-
bolic acidosis based on decreases in blood HCO3− and
base excess concurrent with a decrease in blood pCO2
(Table 1; Figure 1B–D). This resulted in acidemia with
the reduction in DCAD causing a quadratic (P = 0.01)
decline in blood pH (Table 1; Figure 1A). The total
dissolved CO2 in venous blood decreased with DCAD,
and the response tended (P = 0.06) to be quadratic.
Treatment did not affect SO2 in venous blood, hemato-
crit, or the concentration of hemoglobin. As expected,
urinary pH declined with DCAD, and this response
was observed throughout the prepartum period and
throughout the day (Table 1; Figure 2A, B). The inter-
action (P = 0.004) between treatment and day relative
to calving affected urinary pH because the differences
among treatment varied along the prepartum period
mostly because of small changes in pH in cows fed N50
(Figure 2B).
Postpartum Acid-Base Balance
Interactions (P < 0.05) between treatment and day
were observed for pCO2, pO2, and TCO2 postpartum
(Table 2; Figure 1B). The interaction between treat-
ment and day for pCO2 was because on the day of
calving, as the DCAD decreased, so did pCO2, whereas
on d 4 postpartum, the opposite response was observed
(Figure 1B). The same pattern was observed for TCO2.
Saturation of O2 in venous blood tended (P = 0.09) to
increase linearly with a reduction in DCAD (Table 2).
Interactions (P < 0.001) between treatment and day
were observed for blood pH, HCO3-, and base excess
postpartum (Table 2), because the values for all 3 re-
sponses decreased with DCAD on the day of calving,
but the opposite response was observed in the following
days postpartum (Figure 1A, C, D). Treatment did not
affect hematocrit or content of hemoglobin in blood
postpartum.
Prepartum Concentrations of Minerals
and Metabolites in Blood
Concentrations of blood iCa increased linearly (P <
0.001) with a decrease in DCAD (Table 3; Figure 3A);
however, the same decrease in DCAD only tended (P =
0.06) to increase concentration of tCa in serum (Figure
3B). Reducing the DCAD linearly increased (P = 0.04)
the proportion of iCa relative to tCa, and blood iCa
represented 48.8% of the tCa in all prepartum cows.
Treatment did not affect the concentrations of tMg,
tP, and fatty acids in serum or those of iNa and iK in
whole blood. (Table 3; Figures 3C, 3D, and 4B). On the

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 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS

other hand, as the DCAD decreased, concentrations of
glucose in serum prepartum linearly (P < 0.001) de-
creased (Table 3; Figure 4A).
Postpartum Concentrations of Minerals
and Metabolites in Blood
Concentrations of iCa and tCa decreased (P < 0.001)
postpartum reaching their nadir 2 d after calving
(Figure 3A, B). An interaction (P < 0.001) between
treatment and day was observed for blood iCa con-
centrations because they differed among treatments
on the day of calving, but not after that (Figure 3A).
Blood iCa represented 47.4% of the tCa postpartum
and the interaction (P = 0.009) between treatment and
day postpartum affected the ratio because on the day
of calving, reducing the DCAD increased (P = 0.01)
the ratio (P200 = 46.5 vs. N50 = 49.1 vs. N150 = 49.2
± 7.6%), after which the ratios changed and, on d 4
postpartum, they represented 48.5% for P200, 48.4%
for N50, and 46.7% for N150.
Treatment did not affect the concentrations of tCa,
tP, glucose, or fatty acids in serum postpartum (Table
4). Concentrations of tP in serum sharply declined (P

Figure 1. Pre- and postpartum venous blood pH (A), partial pressure of CO2 (pCO2; B), concentration of HCO3− (C), and base excess (D)
in nulliparous cows fed prepartum diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to
calving; after calving, cows were fed the same lactation diet for the first 100 d postpartum. (A) Prepartum: Effects of interaction between treat-
ment and day (P = 0.42), and linear (P < 0.001) and quadratic (P = 0.01) orthogonal polynomial contrasts; postpartum: Effects of interaction
between treatment and day (P < 0.001), and linear (P = 0.03) and quadratic (P = 0.10) orthogonal polynomial contrasts. (B) Prepartum:
Effects of interaction between treatment and day (P = 0.12), and linear (P < 0.001) and quadratic (P = 0.65) orthogonal polynomial contrasts;
postpartum: Effects of interaction between treatment and day (P < 0.001), and linear (P = 0.95) and quadratic (P = 0.74) orthogonal polyno-
mial contrasts. (C) Prepartum: Effects of interaction between treatment and day (P = 0.43), and linear (P < 0.001) and quadratic (P = 0.05)
orthogonal polynomial contrasts; postpartum: Effects of interaction between treatment and day (P < 0.001), and linear (P = 0.05) and quadratic
(P = 0.05) orthogonal polynomial contrasts. (D) Prepartum: Effects of interaction between treatment and day (P = 0.35), and linear (P < 0.001)
and quadratic (P = 0.01) orthogonal polynomial contrasts; postpartum: Effects of interaction between treatment and day (P < 0.001), and linear
(P = 0.01) and quadratic (P = 0.03) orthogonal polynomial contrasts. *Within day, treatments differ (P < 0.05). Error bars represent the SEM.

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 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS

Figure 2. Prepartum urine pH from −21 to −1 d relative to calving (A) and urine pH relative to time of feeding (B) in nulliparous cows
fed prepartum diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calving. (A) Effects
of interaction between treatment and day (P = 0.004), and linear (P < 0.001) and quadratic (P < 0.001) orthogonal polynomial contrasts. (B)
Effects of interaction between treatment and hour (P = 0.71), and linear (P < 0.001) and quadratic (P = 0.07) orthogonal polynomial contrasts.
*Within day, treatments differ (P < 0.001). Error bars represent the SEM.

< 0.001) on the day of calving to a mean of 1.23 mM,
after which it increased to approximately 1.50 mM in
the days following calving (Figure 3D). Concentrations
of tMg in serum increased (P < 0.001) in the first 2 d
postpartum and this increase was greater (P < 0.001) for
cows fed N150 than those fed P200 or N50 (Figure 3C).
After d 2 postpartum, concentrations of tMg decreased
(P < 0.001) and reached a nadir on d 5 postpartum, a
mean concentration similar to that observed during the
prepartum period (0.82 mM). Treatment did not affect
the concentrations of iNa or iK in blood postpartum,
although an effect of day (P < 0.001) was observed for
both minerals in blood because their concentrations de-
creased in blood from calving to d 4 postpartum (iNa,
from 145 to 141 mM; iK from 3.95 to 3.79 mM). A
tendency (P = 0.09) for interaction between treatment
and day was observed for serum BHB concentrations
because they were smaller (P = 0.02) for cows fed N50
than P200 or N150 on d 7 postpartum, after which they
no longer differed (Figure 4C).

Table 2. Effect of DCAD fed prepartum to nulliparous Holstein cows on postpartum measures of blood chemistry and acid-base balance

Treatment2 P-value3

Item1 P200 N50 N150 SEM TRT × day Linear Quadratic

4
Venous blood gas              
 pCO2, mm Hg 41.1 40.9 41.1 0.4 <0.001 0.95 0.74
 pO2, mm Hg 32.7 33.5 34.1 1.2 0.02 0.06 0.80
 TCO2, mM 30.9 30.9 32.0 0.3 <0.001 0.05 0.04
 sO2, % 67.6 68.9 70.1 1.0 0.23 0.09 0.65
Venous blood chemistry              
 pH 7.466 7.467 7.477 0.003 <0.001 0.03 0.10
 HCO3−, mM 29.7 29.7 30.7 0.3 <0.001 0.05 0.05
  Base excess, mM 5.83 5.92 7.18 0.32 <0.001 0.01 0.03
  Hematocrit, % 30.0 29.2 29.9 0.4 0.49 0.59 0.14
  Hemoglobin, g/dL 10.19 9.93 10.16 0.13 0.40 0.58 0.16
1
Blood sampled by puncture of the jugular vein immediately after calving and on d 1, 2, and 4 postpartum from a subset of randomly selected
blocks that had 34, 35, and 36 cows in the P200, N50, and N150 treatment, respectively.
2
Nulliparous cows were fed prepartum diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation
to calving; after calving, cows were fed the same lactation diet for the first 100 d postpartum.
3
TRT × day = interaction between treatment (P200 vs. N50 vs. N150) and day; Linear = linear effect of DCAD; Quadratic = quadratic effect
of DCAD.
4
pCO2 = partial pressure of CO2; pO2 = partial pressure of O2; TCO2 = total dissolved CO2; sO2 = saturation of O2.

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Prepartum Mineral Retention retention of Ca and Mg in the first week of lactation.

Mineral retention was evaluated in the last week of
gestation and cows had been on treatment approxi-
mately 20 d. Treatment did not affect digestibility of
DM or intakes of Ca, Mg, or P (Table 5). Neverthe-
less, reducing the DCAD caused a linear (P < 0.001)
decrease in fecal excretion of Ca because the amount
of Ca apparently absorbed increased 62% in cows fed
N150 compared with P200. At the same time that ap-
parent digestibility of Ca increased, urinary excretion
of Ca also increased (P < 0.001) linearly with a reduc-
tion in DCAD. In spite of the observed changes in Ca
absorption, apparent Ca retention did not change with
treatment, with a mean of 13.9 ± 1.6 g/d. Reducing the
DCAD linearly increased (P = 0.04) the apparent ab-
sorption and digestibility of Mg, whereas it tended (P
= 0.07) to decrease urinary excretion, which resulted
in a linear (P = 0.01) increase in apparent retention of
Mg (Table 5). Reducing the DCAD had a quadratic (P
= 0.04) effect increasing the amount of P apparently
absorbed and cows fed N50 had the largest amount of
P absorbed prepartum.
Postpartum Mineral Retention
Mineral retention was evaluated in the first week
of lactation and apparent digestibility of DM did not
differ among treatments and averaged 74.0 ± 0.9%.
Treatment did not affect the intakes of Ca, P, and Mg
(Table 6). In fact, prepartum DCAD did not affect fe-
cal excretion of those minerals, apparent digestibility
of Ca, Mg, or P, milk secretion of Ca or Mg, or the
Apparent digestibilities of Ca, Mg, and P had means of
43.0 ± 2.7, 45.8 ± 2.6, and 50.0 ± 2.4%, respectively.
The concentrations of Ca and Mg in milk decreased (P
< 0.001) in the first 5 d postpartum (Figure 5A, B).
Similarly, secretion of Mg in milk decreased (P < 0.001)
from d 0 to 5 postpartum, from 3.43 ± 0.09 g/d on the
day of calving to 2.63 ± 0.09 g/d on d 5 (Figure 5D).
Nevertheless, in spite of the concentration of Ca being
greatest in milk on the day of calving, daily secretion
of Ca in milk increased (P < 0.001) from 24.8 ± 0.7
g/d on the day of calving to 31.5 ± 0.7 g/d on d 5
postpartum (Figure 5C).
Incidence of Subclinical Diseases and Hepatic
Tissue Composition
Treatment did not affect the incidence of subclinical
hypocalcemia either based on iCa ≤1.0 mM or serum
tCa ≤2.0 mM (Table 7). Furthermore, treatment did
not affect the incidence of cows with persistent sub-
clinical hypocalcemia or the mean daily prevalence of
subclinical hypocalcemia in the first 7 d postpartum.
Treatment did not influence the incidence of hyperke-
tonemia that affected 33.3%; however, as the DCAD
decreased, the mean daily prevalence of hyperketone-
mia also decreased (P = 0.04; Table 7). Treatment did
not affect the concentration of triacylglycerol in the
hepatic tissue on d 10 postpartum either on wet or DM
basis. Treatment did not affect the prevalence of fatty
liver, based on hepatic triacylglycerol >5.0% on a wet
basis, which affected 51% of the cows. The rectal tem-
perature of cows tended (P = 0.08) to responded qua-

Table 3. Effect of DCAD fed prepartum to nulliparous Holstein cows on prepartum concentrations of minerals and metabolites

Treatment1 P-value2

Item P200 N50 N150 SEM TRT × day Linear Quadratic

3
Blood ionized Ca, mM 1.224 1.243 1.259 0.008 0.29 <0.001 0.42
Ionized Ca, % tCa 47.8 49.4 49.1 0.7 0.76 0.04 0.28
Serum tCa,4 mM 2.50 2.53 2.56 0.02 0.14 0.06 0.66
Serum tMg,4 mM 0.82 0.80 0.82 0.01 0.99 0.66 0.13
Serum tP,4 mM 1.71 1.71 1.73 0.03 0.88 0.69 0.66
Blood ionized Na,3 mM 143.3 143.3 143.8 0.2 0.92 0.23 0.21
Blood ionized K,3 mM 3.80 3.88 3.85 0.03 0.52 0.14 0.32
Serum glucose,5 mM 4.69 4.35 4.36 0.08 — 0.001 0.30
Serum fatty acids,5 mM 0.45 0.49 0.45 0.03 — 0.74 0.33
1
Nulliparous cows were fed prepartum diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation
to calving; after calving, cows were fed the same lactation diet for the first 100 d postpartum.
2
TRT × day = interaction between treatment (P200 vs. N50 vs. N150) and day; Linear = linear effect of DCAD; Quadratic = quadratic effect
of DCAD.
3
Blood sampled by puncture of the jugular vein on days −6 and −3 relative to calving from a subset of randomly selected blocks that had 28,
32, and 31 cows in the P200, N50, and N150 treatment, respectively.
4
Blood sampled by puncture of coccygeal vessels from all cows on days −9, −7, −5, −3, and −1 relative to calving.
5
Blood sampled by puncture of coccygeal vessels from all cows on day −7 relative to calving.

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 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS

dratically to prepartum DCAD, but differences were of
small magnitude and no interaction (P = 0.25) between
treatment and day postpartum. Nevertheless, both the
incidence (P = 0.03) and the mean daily prevalence of
fever (P = 0.02) decreased in a quadratic fashion in
response to prepartum DCAD (Table 7).
Incidence of Clinical Diseases and Survival
No case of clinical hypocalcemia was diagnosed in
the experiment. Retained placenta affected 13.6% of
the cows and prepartum DCAD tended (P = 0.08)
to have a quadratic effect because it was lowest in
cows fed N50 (Table 8). Although treatment did not
affect the incidence of metritis or puerperal metritis,
the incidence of uterine disease, which combined re-
tained placenta and metritis, tended (P = 0.06) to be
affected quadratically by prepartum DCAD. Reducing
the DCAD from P200 to N50 reduced the incidence
of uterine disease, but further reduction in DCAD
caused an increase in the incidence of uterine disease.
Mastitis and displaced abomasum affected each 4.6%

Figure 3. Pre- and postpartum concentrations of blood ionized Ca (A), and serum total Ca (B), serum total Mg (C), and serum total P (D)
in nulliparous cows fed prepartum diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to
calving; after calving, cows were fed the same lactation diet for the first 100 d postpartum. (A) Prepartum: Effects interaction between treat-
ment and day (P = 0.29), and linear (P < 0.001) and quadratic (P = 0.42) orthogonal polynomial contrasts; postpartum: Effects of interaction
between treatment and day (P < 0.001), and linear (P = 0.97) and quadratic (P = 0.87) orthogonal polynomial contrasts. (B) Prepartum:
Effects of interaction between treatment and day (P = 0.14), and linear (P = 0.06) and quadratic (P = 0.66) orthogonal polynomial contrasts;
postpartum: Effects of interaction between treatment and day (P = 0.33), and linear (P = 0.78) and quadratic (P = 0.62) orthogonal polynomial
contrasts. (C) Prepartum: Effects of interaction between treatment and day (P = 0.99), and linear (P = 0.66) and quadratic (P = 0.13) orthogo-
nal polynomial contrasts; postpartum: Effects of interaction between treatment and day (P = 0.002), and linear (P = 0.03) and quadratic (P =
0.03) orthogonal polynomial contrasts. (D) Prepartum: Effects of interaction between treatment and day (P = 0.88), and linear (P = 0.69) and
quadratic (P = 0.66) orthogonal polynomial contrasts; postpartum: Effects of interaction between treatment and day (P = 0.36), and linear (P
= 0.90) and quadratic (P = 0.55) orthogonal polynomial contrasts. *Within day, treatments differ (P < 0.01). Error bars represent the SEM.

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 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS

Figure 4. Pre- and postpartum concentrations of glucose (A), fatty acids (B), and BHB (C) in serum of nulliparous cows fed prepartum
diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calving; after calving, cows were fed
the same lactation diet for the first 100 d postpartum. Panel A. Prepartum: Effects of linear (P = 0.001) and quadratic (P = 0.30) orthogonal
polynomial contrasts; Postpartum: Effects of interaction between treatment and day (P = 0.77), and linear (P = 0.38) and quadratic (P = 0.57)
orthogonal polynomial contrasts. Panel B. Prepartum: Effects of linear (P = 0.74) and quadratic (P = 0.33) orthogonal polynomial contrasts;
Postpartum: Effects of interaction between treatment and day (P = 0.60), and linear (P = 0.73) and quadratic (P = 0.38) orthogonal polynomial
contrasts. Panel C. Effects of interaction between treatment and day (P = 0.09), and linear (P = 0.17) and quadratic (P = 0.21) orthogonal
polynomial contrasts. * Within day, treatments differ (P = 0.02). Error bars represent the SEM.

of the cows in the experiment and treatment did not
affect the risk of mastitis; however, treatment influ-
enced (P = 0.02) the risk of displaced abomasum
because the incidence was 0 in cows fed N50 and it
was 11.4% in cows fed N150. Because of the responses
of individual diseases, treatment resulted in quadratic
(P < 0.03) effects on morbidity and on risk of cows
having multiple diseases in the first 100 d postpar-
tum (Table 8). Overall, both morbidity and multiple
diseases was smallest in cows fed N50 and greatest in
cows fed N150. A tendency (P = 0.07) for a linear re-
duction in removal from the herd was observed as the
prepartum DCAD decreased (Table 8). Of the cows
that left the herd by 305 d postpartum, 5 either died
or were euthanized (3 P200: 1 with peritonitis, 1 with
lymphosarcoma, and 1 with broken leg; 2 N50: 1 with
lymphosarcoma and 1 with dislocated hip joint), and
5 were sold (3 P200, 1 N50, 1 N150).

Table 4. Effect of DCAD fed prepartum to nulliparous Holstein cows on postpartum concentrations of minerals and metabolites

Treatment1 P-value2

Item P200 N50 N150 SEM TRT × day Linear Quadratic

3
Blood ionized Ca, mM 1.122 1.121 1.123 0.008 <0.001 0.97 0.87
Ionized Ca, % tCa 47.5 47.7 47.0 0.5 0.009 0.57 0.35
Serum tCa,4 mM 2.38 2.37 2.38 0.02 0.33 0.78 0.62
Serum tMg,4 mM 0.85 0.85 0.89 0.01 0.002 0.03 0.03
Serum tP,4 mM 1.44 1.43 1.44 0.02 0.36 0.90 0.55
Blood ionized Na,3 mM 142.9 143.1 143.2 0.2 0.37 0.28 0.98
Blood ionized K,3 mM 3.87 3.86 3.83 0.03 0.24 0.36 0.65
Serum glucose,5 mM 4.30 4.39 4.36 0.07 0.77 0.38 0.57
Serum fatty acids,6 mM 0.82 0.78 0.81 0.03 0.60 0.73 0.38
Serum BHB,7 mM 0.87 0.78 0.82 0.02 0.09 0.17 0.21
1
Nulliparous cows were fed prepartum diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation
to calving; after calving, cows were fed the same lactation diet for the first 100 d postpartum.
2
TRT × day = interaction between treatment (P200 vs. N50 vs. N150) and day; Linear = linear effect of DCAD; Quadratic = quadratic effect
of DCAD.
3
Blood sampled by puncture of the jugular vein immediately after calving and on d 1, 2, and 4 postpartum from a subset of randomly selected
blocks that had 34, 35, and 36 cows in the P200, N50, and N150 treatment, respectively.
4
Blood sampled by puncture of coccygeal vessels from all cows on d 1, 2, 3, 4, 5, and 7 postpartum.
5
Blood sampled by puncture of coccygeal vessels from all cows on d 0, 1, 2, 3, 4, 5, 7, 14, and 21.
6
Blood sampled by puncture of coccygeal vessels from all cows on d 0, 7, 14, and 21.
7
Blood sampled by puncture of coccygeal vessels from all cows on d 7, 14 and 21.

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Table 5. Effect of DCAD fed prepartum to nulliparous Holstein cows on intake, excretion, and retention of minerals in the last week of gestation

Treatment2 P-value3

Item1 P200 N50 N150 SEM Linear Quadratic

First day of sampling relative to calving −4.7 −4.8 −5.1 0.6 0.71 0.79
Days on treatment at first day of sampling 20.2 19.7 19.8 0.4 0.45 0.74
DMI, kg/d 8.09 8.13 7.98 0.26 0.85 0.68
DM apparent digestibility, % 62.5 63.7 63.8 1.1 0.40 0.84
Calcium            
  Intake, g/d 54.0 55.0 51.8 1.8 0.54 0.20
  Fecal excretion, g/d 41.0 36.0 31.0 1.6 <0.001 0.22
  Apparent absorption, g/d 12.9 19.0 20.9 1.4 <0.001 0.86
  Apparent digestibility, % 24.1 34.0 40.7 2.2 <0.001 0.43
  Urinary excretion, g/d 0.17 2.81 6.90 0.44 <0.001 0.44
  Apparent retention, g/d 12.6 15.6 13.4 1.6 0.48 0.19
Magnesium            
  Intake, g/d 35.2 37.0 36.5 1.3 0.37 0.57
  Fecal excretion, g/d 28.1 27.1 26.0 1.5 0.30 0.75
  Apparent absorption, g/d 7.04 9.86 10.40 1.26 0.04 0.80
  Apparent digestibility, % 20.1 25.6 29.0 3.1 0.04 0.78
  Urinary excretion, g/d 6.45 5.14 5.60 0.40 0.07 0.17
  Apparent retention, g/d 0.56 4.76 4.69 1.22 0.01 0.41
Phosphorus            
  Intake, g/d  27.4 29.3 27.9 1.0 0.47 0.19
  Fecal excretion, g/d 17.7 18.0 18.8 1.0 0.42 0.65
  Apparent absorption, g/d 9.83 11.35 9.16 0.85 0.92 0.04
  Apparent digestibility, % 35.7 38.2 33.0 2.5 0.66 0.12
1
Samples collected on 2 consecutive days prepartum, at 270 and 271 of gestation, from a subset of randomly selected blocks that had 26, 30, and
29 cows in the P200, N50, and N150 treatment, respectively.
2
Nulliparous cows were fed prepartum diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation
to calving; after calving, cows were fed the same lactation diet for the first 100 d postpartum.
3
Linear = linear effect of DCAD; Quadratic = quadratic effect of DCAD.

DISCUSSION Changes in mineral metabolism were observed with

Limited data exist investigating the effects of ma-
nipulating the DCAD of prepartum diets on metabo-
lism and health of nulliparous cows (Lean et al., 2019;
Santos et al., 2019). To a large extent, the scarcity of
data is justified by the fact that cows starting their first
lactation almost never are diagnosed with clinical hy-
pocalcemia (DeGaris and Lean, 2008; Lean et al., 2019;
Santos et al., 2019), and the prevalence of subclinical
hypocalcemia usually is less than half of that observed
in parous cows (Reinhardt et al., 2011; Neves et al.,
2018). As shown by others, no case of clinical hypocal-
cemia was observed in cows starting their first lactation
in the present experiment (Santos et al., 2019), and
incidence and daily prevalence of subclinical hypocal-
cemia were substantially less than values reported in
the literature for parous cows (Reinhardt et al., 2011;
Neves et al., 2018). Nevertheless, some indication exists
that reducing the DCAD in prepartum diets might ben-
efit health of nulliparous cows (Lean et al., 2019; Santos
et al., 2019). In fact, results from the present experi-
ment suggest that a moderate acidogenic diet reduced
fever, morbidity, and risk of multiple diseases to a large
extent because of a reduction in uterine diseases.
manipulating the DCAD prepartum. As the DCAD
decreased, concentrations of iCa in blood prepartum
and on the day of calving and those of tMg in serum
postpartum increased. The increase in concentrations of
iCa in blood prepartum with a reduction in DCAD was
anticipated because consuming acidogenic diets induce
a state of compensated metabolic acidosis, as observed
in the present experiment, which typically displaces Ca
bound to albumin or salts in blood (Oberleithner et al.,
1982), thus resulting in ionization of Ca that causes
a shift in the ratio of iCa to tCa (Vieira-Neto et al.,
2021b). Such responses with increased proportion of
tCa as iCa were observed herein in the days preceding
and on the day of calving, but not afterward. These
results corroborate previously published data with nul-
liparous and parous cows that acidogenic diets increase
concentrations of iCa and the proportion of tCa in blood
in the ionized form (Rodney et al., 2018; Vieira-Neto et
al., 2021a). The treatments implemented also affected
the concentrations of tMg in the first days postpartum.
Concentrations of tMg typically increase in blood as
those of iCa decrease with the onset of lactation (Lop-
era et al., 2018; Rodney et al., 2018), and these changes
are thought to be mediated by renal handling of Mg.

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 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS

Changes in response of Ca-sensing receptors induced by
the reduced concentration of iCa in the thick ascending
limb of the Henle loop and distal convoluted tubules
stimulates the Na+/K+/2Cl− cotransporter system,
which is important for paracellular reabsorption of iCa
and Mg2+ (Dai et al., 2001). The expected changes in
the Na+/K+/2Cl− cotransporter system induced by
the reduced concentration of iCa in the days following
calving cause an increase in the lumen-positive voltage,
which increases the paracellular reabsorption of Mg2+
from the glomerular filtrate (Hebert et al., 1997), which
is expected to conserve Mg. Cows fed the acidogenic
diets underwent a more sudden change in acid-base bal-
ance once lactation started and the ratio of iCa to tCa
shifted after d 0, which might have influenced the pH
and content of iCa in the urinary filtrate. The increase
in blood pH and concentrations of HCO3- postpartum
which might have alkalinized the urinary filtrate and
increased the renal reabsorption of iCa through the
transient receptor potential cation channel subfamily
V member 5 (Yeh et al., 2003), thus reducing the ex-
posure of the distal convoluted tubules to iCa, which
should reduce loss of Mg2+ in the urine (Hebert et al.,
1997). Also, cows fed the acidogenic diets had increased
Mg retention prepartum, which might have increased
labile Mg stores and contributed to the differences in
serum concentrations of tMg in early lactation.
Acidogenic diets induced the typical changes in blood
gases and acid-base balance prepartum. Cows fed N50
and N150 experienced a moderate decline in blood
pH, reduced concentrations of blood bicarbonate and
base excess, increased net acid urinary excretion, and
reduced blood pCO2, all hallmarks of acidemia con-
current with metabolic acidosis (Charbonneau et al.,
2006). Nevertheless, it is interesting to note that the
opposite responses to prepartum DCAD were observed
postpartum with a more pronounced increase in blood
pH, pCO2, HCO3- and base excess in cows fed N150
compared with those fed P200. A similar response has
previously been observed in dairy cows fed prepartum
diets with diverging DCAD in which responses to
DCAD shift in opposite directions between the pre- and
postpartum periods (Rodney et al., 2018). It is possible
that respiratory compensatory mechanisms to induce
hypocapnia with increased respiratory frequency dur-
ing metabolic acidosis (Zimpel et al., 2018), and the
increased urinary excretion of protons as ammonium
ion as cows consume diets with more strong anions
(Constable et al., 2019), might cause a faster response
to a shift to an alkalogenic fed immediately after calv-

Table 6. Effect of DCAD fed prepartum to nulliparous Holstein cows on intake, excretion, and retention of minerals in the first week postpartum

Treatment2 P-value3

Item1 P200 N50 N150 SEM Linear Quadratic

DMI, kg/d 12.4 12.4 12.5 0.4 0.90 0.97
DM apparent digestibility, % 73.9 74.2 74.0 0.9 0.87 0.79
Calcium            
  Intake, g/d 78.8 79.6 78.6 2.2 0.98 0.72
  Fecal excretion, g/d 46.4 46.3 44.2 2.7 0.59 0.62
  Apparent absorption, g/d 32.4 33.3 34.3 2.1 0.49 0.85
  Apparent digestibility, % 42.5 42.4 44.1 2.7 0.71 0.66
  Urinary excretion, g/d 0.105 0.106 0.092 0.008 0.18 0.13
  Milk secretion, g/d 31.3 29.2 30.0 0.9 0.22 0.33
  Apparent retention, g/d 1.05 3.93 4.21 2.31 0.26 0.82
Magnesium            
  Intake, g/d 61.9 63.8 63.3 1.9 0.45 0.66
  Fecal excretion, g/d 34.2 34.4 34.5 2.0 0.88 0.99
  Apparent absorption, g/d 27.8 29.4 28.9 1.7 0.47 0.64
  Apparent digestibility, % 45.7 45.9 45.8 2.6 0.98 0.95
  Urinary excretion, g/d 5.28 5.33 5.07 0.31 0.71 0.59
  Milk secretion, g/d 2.66 2.55 2.62 0.08 0.51 0.42
  Apparent retention, g/d 19.9 21.5 21.2 1.7 0.42 0.70
Phosphorus            
  Intake, g/d 47.4 47.8 47.2 1.4 0.99 0.72
  Fecal excretion, g/d 23.7 23.3 24.8 1.4 0.68 0.45
  Apparent absorption, g/d 23.7 24.5 22.4 1.3 0.64 0.22
  Apparent digestibility, % 50.7 51.4 47.9 2.4 0.50 0.31
1
Samples collected on 2 consecutive days postpartum, 5 and 6, from a subset of randomly selected blocks that had 35, 39, and 34 cows in the
P200, N50, and N150 treatment, respectively.
2
Nulliparous cows were fed prepartum diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation
to calving; after calving, cows were fed the same lactation diet for the first 100 d postpartum.
3
Linear = linear effect of DCAD; Quadratic = quadratic effect of DCAD.

Journal of Dairy Science Vol. 104 No. 12, 2021

 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS

Figure 5. Concentrations of Ca (A) and Mg (B) and secretions of Ca (C) and Mg (D) in milk of nulliparous cows fed prepartum diets with
a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calving; after calving, cows were fed the same
lactation diet for the first 100 d postpartum. Panel A: Effects of interaction between treatment and day (P = 0.57), and linear (P = 0.82) and
quadratic (P = 0.53) orthogonal polynomial contrasts. Panel B: Effects of interaction between treatment and day (P = 0.53), and linear (P =
0.69) and quadratic (P = 0.54) orthogonal polynomial contrasts. Panel C: Effects of interaction between treatment and day (P = 0.94), and
linear (P = 0.12) and quadratic (P = 0.61) orthogonal polynomial contrasts. Panel D: Effects interaction between treatment and day (P = 0.86),
and linear (P = 0.24) and quadratic (P = 0.47) orthogonal polynomial contrasts. Error bars represent the SEM.

ing with a more pronounced alkalinization of blood
and changes in blood gases and measures of acid-base
balance. It is important to note that time relative to
feeding did not affect urinary pH, thus suggesting that
for cows receiving acidogenic diets as TMR, sampling
time to monitor urinary pH is not critical. In fact, these
results corroborate those of Vieira-Neto et al. (2021b)
who also showed that urinary pH remained constant
throughout the day after cows had received the acido-
genic diet for at least 36 h.
Feeding acidogenic diets have been shown to enhance
tissue response to parathyroid hormone (PTH) in dairy
cows (Goff et al., 2014; Vieira-Neto et al., 2021b), and
metabolic acidosis has been shown to directly stimu-

Journal of Dairy Science Vol. 104 No. 12, 2021

 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS

Table 7. Effect of DCAD fed prepartum to nulliparous Holstein cows on subclinical diseases, hepatic composition, and rectal temperature

Treatment1 P-value2

Item Incidence (n/n) P200 N50 N150 SEM Linear Quadratic

Incidence of hypocalcemia              
  iCa ≤1.0 mM,3 % 14.7 (15/102) 14.4 18.0 10.4 6.3 0.75 0.40
  tCa ≤2.0 mM,4 % 26.5 (35/132) 33.3 21.3 23.9 7.0 0.70 0.47
 Persistent5 10.6 (14/132) 13.5 4.1 12.6 4.6 0.50 0.13
  Mean daily prevalence4 — 6.6 3.5 5.1 1.6 0.33 0.28
Hyperketonemia,6 %              
 Incidence 33.3 (44/132) 38.5 35.4 27.1 7.3 0.32 0.55
  Mean daily prevalence — 19.7 13.7 9.4 3.5 0.04 0.58
Hepatic composition7              
  DM, % — 34.60 35.41 34.30 0.59 0.98 0.17
  Triacylglycerol, % as is — 5.11 5.80 4.94 0.49 0.94 0.18
  Triacylglycerol, % of DM — 14.71 16.35 14.52 1.39 0.86 0.28
  Fatty liver,7 % 51.0 (50/98) 52.0 58.4 43.2 9.7 0.68 0.27
  Rectal temperature,8 °C — 38.76 38.70 38.77 0.03 0.76 0.08
Fever,9 %              
 Incidence 32.6 (43/132) 46.5 17.6 33.9 7.0 0.06 0.03
  Daily prevalence — 5.6 2.0 4.5 1.0 0.11 0.02
1
Nulliparous cows were fed prepartum diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation
to calving; after calving, cows were fed the same lactation diet for the first 100 d postpartum.
2
Linear = linear effect of DCAD; Quadratic = quadratic effect of DCAD.
3
Blood sample with ionized Ca (iCa) ≤ 1.0 mM on d 0, 1, 2, or 4 postpartum.
4
Serum sample with total Ca (tCa) ≤ 2.0 mM on d 0, 1, 2, 3, 4, 5, or 7 postpartum.
5
At least 2 serum samples with tCa ≤2.0 mM on d 0, 1, 2, 3, 4, 5, or 7 postpartum.
6
Serum sample with BHB >1.2 mM on d 7, 14, or 21 d postpartum.
7
Hepatic tissue sampled on d 10 ± 1 postpartum. Fatty liver based on triacylglycerol >5% on as is basis.
8
Rectal temperature measured between 0800 and 1000 h on d 1, 2, 3, 4, 5, 7, 9, 11, 13, and 15 postpartum. Interaction between treatment and
day (P = 0.25).
9
Rectal temperature >39.5°C on d 1, 2, 3, 4, 5, 7, 9, 11, 13, or 15 postpartum.

late PTH secretion by the parathyroid gland (Lopez (Vieira-Neto et al., 2017), which acts in the enterocytes
et al., 2002). Among the numerous effects of PTH, it enhancing the expression of the apical membrane Ca
induces synthesis of 1,25-dihydroxyvitamin D3 (Goff et transporter, transient receptor potential cation chan-
al., 2014), a potent calciotropic hormone in dairy cows nels vanilloid 6 (Van Cromphaut et al., 2001), the

Table 8. Effect of dietary cation-anion difference fed prepartum to nulliparous Holstein cows on incidence of diseases and survival

Treatment2 P-value3

Item1 Incidence (n/n) P200 N50 N150 SEM Linear Quadratic

Retained placenta, % 13.6 (18/132) 14.5 6.7 20.4 5.2 0.89 0.08
Metritis, % 34.1 (45/132) 35.7 25.4 41.1 7.3 0.93 0.13
Puerperal metritis, % 9.9 (13/132) 5.0 5.0 12.9 4.0 0.30 0.32
Uterine disease,4 % 35.6 (47/132) 36.3 25.6 46.0 7.3 0.68 0.06
Mastitis,5 % 4.6 (6/132) 0 4.4 6.8 — — —
Displaced abomasum,5 % 4.6 (6/132) 2.4 0.0 11.4 — — —
Morbidity, % 40.9 (54/132) 41.4 28.1 55.6 7.3 0.48 0.02
Multiple diseases, % 18.2 (24/132) 16.3 8.9 29.6 5.8 0.45 0.03
Left the herd,6 % 7.6 (10/132) 13.7 6.7 2.3 3.8 0.07 0.53
1
Incidence of diseases collected until 100 d postpartum and survival until 305 DIM.
2
Nulliparous cows were fed prepartum diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation
to calving; after calving, cows were fed the same lactation diet for the first 100 d postpartum.
3
Linear = linear effect of DCAD; Quadratic = quadratic effect of DCAD.
4
Cows that had retained fetal membranes or metritis.
5
Analyzed by the Fisher’s exact test because of the low frequency of events. Effect of treatment on risk of mastitis (P = 0.32) and displaced
abomasum (P = 0.02).
6
Sold or dead by 305 DIM.

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 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS
basolateral Ca transporters, plasma membrane Ca
ATPase, and the Na/Ca exchanger type 1 (Lee et al.,
2015). These actions are expected to increase gastro-
intestinal absorption of Ca. Indeed, as the prepartum
DCAD decreased, apparent digestibility of Ca and
the amount of Ca absorbed linearly increased, which
corroborates results from Vieira-Neto et al. (2021b)
showing a stimulatory effect of acidogenic diets on Ca
absorption. In the bovine, most Ca absorption takes
place before the duodenum (Khorasani and Armstrong,
1992), presumably in the rumen-reticulum. Schröder et
al. (2015) demonstrated the presence of transcellular
transport of Ca in the bovine ruminal epithelia and
feeding acidogenic diets to sheep increased the flux of
Ca from the mucosal to the serosal side in ruminal epi-
thelia (Wilkens et al., 2016). In spite of the additional
16-percentage unit increase in apparent digestibility of
Ca in cows fed N150 compared with P200, retention
of Ca did not change with treatment because the ac-
idogenic diets not only increased absorption, but they
also increased renal Ca excretion (Vieira-Neto et al.,
2021b). Apparent digestibility of Mg also increased
with a reduction in DCAD, a finding that had not been
previously observed (Vieira-Neto et al., 2021b). Diets
were formulated to contain the same concentration of
Mg prepartum and adjustments were made in dietary
ingredients to adjust the Mg content by adding Mg as
oxide in treatments P200 and N50 (Supplemental Table
S1, https:​/​/​figshare​.com/​s/​23982480396e0155e6bd).
By contrast, the acidogenic supplement fed prepartum
contains Mg in a chloride form, which might be more
soluble in the rumen, thus facilitating absorption. The
effects of treatment on Ca and Mg retention were ob-
served only prepartum, when differences in acid-base
balance were clear among treatments. Those effects of
treatment on mineral retention were no longer observed
postpartum. It is important to note that cows were in
positive Ca and Mg retention prepartum, and likely also
P, although urinary losses of the latter mineral were not
quantified pre- or postpartum. To our surprise, in spite
of the limited DM intake in the first days postpartum,
only 12.4 kg/d, which resulted in Ca intake of 79 g/d,
and the extensive loss of Ca in milk, more than 30 g/d,
cows in the first week of lactation maintained a positive
Ca retention with apparent retention slightly above 0.
It seems that nulliparous cows were able to maintain
a positive Ca retention even in early lactation when
they experienced pronounced negative energy balance
(Zimpel et al., 2021).
Reducing the DCAD in diets of nulliparous cows did
not affect postpartum productive performance pre-
sented in the companion paper (Zimpel et al., 2021),
corroborating previous results (Moore et al., 2000; Mar-
Journal of Dairy Science Vol. 104 No. 12, 2021
tinez et al., 2018). On the other hand, we observed evi-
dence of reduced uterine diseases, morbidity, and risk of
multiple diseases as the DCAD decreased to −50 mEq/
kg in the present experiment. It is important to note
that most disease responses affected by dietary treat-
ments were quadratic, and the benefits were no longer
observed once the DCAD further decreased to −150
mEq/k. Obviously, the present experiment does not
allow us to identify an optimum prepartum DCAD to
reduce risk of diseases, but it reinforces the importance
of avoiding diet-induced excessive metabolic acidifica-
tion in nulliparous cows. Also, the present experiment
enrolled 132 cows, which limits the ability to detect
statistical effects for health data when the observed
differences between treatment are of small magnitude.
Thus, lack of statistical effect should not be interpreted
as evidence of absence of effect.
Two recent meta-analysis of the published literature
(Lean et al., 2019; Santos et al., 2019) observed benefits
of reducing the DCAD to health of dairy cows and the
responses were common to nulliparous and parous cows.
For parous cows, as expected, the reduction in DCAD
markedly reduced the risk of clinical hypocalcemia.
Beyond that, the authors also observed that reducing
the DCAD reduced the risk of retained placenta and
metritis (Lean et al., 2019; Santos et al., 2019). The
justification for reduced risk of diseases was the im-
proved peripartum calcemia, which is linked to uterine
and other diseases (Martinez et al., 2012), in part medi-
ated by changes in innate immune responses (Kimura
et al., 2006; Martinez et al., 2014). Nulliparous cows
fed the acidogenic diets had increased concentrations of
iCa in blood prepartum and on the day of calving, but
those changes did not affect the incidence or the mean
daily prevalence of subclinical hypocalcemia. Neverthe-
less, as the DCAD decreased, the incidence of fever and
the mean daily prevalence of fever in early lactation
decreased in a quadratic manner, likely because of the
reduced morbidity and, perhaps, less severe diseases.
The reduction in DCAD also reduced the mean preva-
lence of hyperketonemia, likely linked to the reduced
risk of clinical diseases in N50. Disease is known to
cause hypophagia and increase tissue catabolism, which
are expected to increase concentration of BHB and the
risk of hyperketonemia. The responses in health herein
and those of reproduction, in which reducing the DCAD
linearly increased the proportion of pregnant cows by
305 DIM, reported in the companion paper (Zimpel
et al., 2021) likely explain the tendency to increase
survival of cows. Collectively, these results corroborate
those observed by Lean et al. (2019) and Santos et
al. (2019) that manipulating the DCAD of prepartum
diets influences health. Furthermore, the observation
 Zimpel et al.: PREPARTUM DCAD FOR NULLIPAROUS COWS
that a moderately acidogenic diet is preferred and that
further decreasing the DCAD to −150 mEq/kg, often
recommended to parous cows, abolished the benefits
observed on morbidity or risk of multiple diseases.
Possible reasons for lack of benefit or even a potential
detrimental effect of excessively acidogenic diets on
health of nulliparous cows might involve the metabolic
acidosis-induced decline in prepartum DMI (Zimpel et
al., 2018), which reduces nutrient balance as observed
in the present experiment (Zimpel et al., 2021). It is
possible that the further reduction in prepartum DM
and nutrient intake in cows fed N150 affected nutrient
supply to the point to increase the risk of uterine dis-
eases. Reduced intake prepartum has been associated
with increased risk of uterine diseases (Pérez-Báez et
al., 2019). Moreover, metabolic acidosis is known to
interfere with tissue metabolism, in particular to affect
insulin release by the pancreas and tissue responsive-
ness to insulin (Bigner et al., 1996; Vieira-Neto et al.,
2021a). As the diet becomes more acidogenic, adipose
tissue responses to insulin shift favoring lipolysis over
lipogenesis (Vieira-Neto et al., 2021a). Thus, it is pos-
sible that exacerbated metabolic acidosis induced by
diets such as that fed in N150 to cows with low risk of
hypocalcemia might be detrimental to health and not
benefit production performance (Zimpel et al., 2021).
CONCLUSIONS
Manipulating the DCAD of prepartum diets altered
acid-base balance and mineral metabolism, which af-
fected health of cows. The reduction in DCAD induced
a state of compensated metabolic acidosis that trig-
gered increased apparent gastrointestinal absorption
and blood concentration of Ca, concurrent with in-
creased renal disposal of Ca. The moderate acidogenic
diet promoted a reduction in the prevalence of fever,
morbidity, and risk of multiple diseases, mostly medi-
ated by reduced risk of uterine diseases. Nevertheless,
the benefits to health were abolished once the DCAD
was further reduced to −150 mEq/kg. The present ex-
periment enrolled 132 nulliparous cows, which limits
the ability to detect statistical effects when the differ-
ence between treatments is of small magnitude. Thus,
lack of statistical effect should not be interpreted as
evidence of absence of effect. Altogether, the current
experiment reinforces previous findings in which ma-
nipulating the prepartum DCAD affects health of nul-
liparous cows; however, it also shows the importance of
avoiding metabolic over-acidification, as this abolished
most of the benefits observed in reduction of clinical
diseases when feeding acidogenic diets.
Journal of Dairy Science Vol. 104 No. 12, 2021
ACKNOWLEDGMENTS
The authors thank Iago M. R. Leão, Jessica G. Prim,
Karla Ferreira, Félix Welter, Ingrid N. F. Edelhoff,
Vitória F. S. de Camargo, Túlio H. Souza, Gustavo
Avelin, Silvia Rodrigues, Gabriel Godoy, and Xun Cao
(University of Florida) for their help ensuring proper
daily care of cows and collection of samples. The help
of the staff of the University of Florida Dairy Unit
(Hague, FL) is greatly appreciated. The authors thank
Elliot Block of Arm and Hammer Animal and Food
Production (Princeton, NJ) for providing Bio-Chlor,
sodium bicarbonate, and potassium carbonate; Terry
Creel and Tim Brown of Landus Cooperative (Ames,
IA) for providing SoyPlus; and Barbara Barton and
Marcos Zenobi of Balchem Animal Nutrition and
Health (New Hampton, NY) for providing ReaShure
for this experiment. Partial funding for this project was
provided by a grant from the Southeast Milk Check-Off
Program (Southeast Milk Inc., Belleview, FL) and by
Arm and Hammer Animal and Food Production. The
authors have not stated any conflicts of interest.
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